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Jiang Qiang
Department of Forensic Medicine
Forensic DNA Typing
Outline
1. Introduction
2. Application of forensic DNA typing
3. Process of forensic DNA typing
4. Conclusion
Brief History of Forensic DNA Typing
• 1980 - Ray White describes first polymorphic RFLP marker
• 1985 - Alec Jeffreys discovers multilocus VNTR probes
• 1985 - first paper on PCR• 1991 - first STR paper• 1995 - UK DNA database• 1998 – US CODIS database• 2000 – Other countries database
• DNA parting standardization
Examples of DNA in the News
2001 “911”, New York
2004Indian OceanTsunami
2008 Wenchuan Earthquake
What are we doing
IdentificationIdentification Paternity testingPaternity testing
How to do
• DNA analysis
• STR,Y-STR,X-STR
• mtDNA,SNP
Fingerprints have been used since 1901
Methods for Human Identification
DNA Fingerprints since 1985
Applications for Forensic DNA Typing
Forensic cases - matching suspect with evidence
Paternity testing - identifying father
Historical investigations
Missing persons investigations
Mass disasters - putting pieces back together
DNA database
Two main tasks of Forensic Biology
Individual Identification Paternity Testing
Polymerase Chain Reaction (PCR)
Locus
Allele
STR
Homozygote
Heterozygote
Basic Concepts
DNA in the Cell
chromosome
cell nucleus
Double stranded DNA molecule
Individual nucleotides
P C R( Polymerase Chain Reaction)
类似于 DNA的天然复制过程,在体外试管内,以一对特异性的寡核苷酸为引物,四种脱氧核苷酸为底物,待测 DNA为模板,在 DNA聚合酶的催化作用下,通过高温变性——低温退火——中温延伸的温度循环,选择性扩增特异性目的 DNA片段过程 。
1 2 3 4 5
22
55
72
94
时间( min )
温度
(℃)
适温延伸3
高温变性1
低温退火2
重复 1 ~ 3 步25 ~ 30 轮
目的 DNA 片段扩增 100 万倍以上
DNA 双螺旋
DNA 单链与引物复性
DN
A
变性
形成2
条单
链 子链延伸DNA 加倍
principle
Father’s Sperm
Mother’s Egg
Child’s Cell
For each inherited characteristic, an organism has two genes, one from each parent. We call these genes alleles.
基因座 (locus)
基因在染色体上的特定位置称基因座。
等位基因 (allele)
同一个基因座上的基因可以有多个,它们之间存在 DNA一级结构差异,导致产生相对性状,这种有差异的基因互称为等位基因。
A
B
个体 1 个体 2
-
+
Short Tandem Repeats (STRs)
AATG
7 repeats
8 repeats
AATG
Homozygote: Alleles are identical on each chromosome ; both alleles are the same length
Heterozygote: Alleles differ on each on each chromosome ; alleles differ and can be resolved from one another
Fluorescent dye label
Fluorescent dye creates a labeled PCR product
Fluorescent dye creates a labeled PCR product
primer1
primer2primer1
primer2
number of repeat units
Locus is the region of the genome being examined
Sources of Biological EvidenceBlood/Blood stain
Semen/ Semen stain
Saliva/Saliva stain
Urine
Hair
Teeth
Bone
Tissue
Blood stainOnly a very small amount of sample is needed to obtain
a DNA profile
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539 D18S51
D8S1179
D3S1358
FGA
VWA
13 CODIS Core STR Loci
AMEL
AMEL
Sex-typing
Forensic STR Markers on Human Chromosomes
Sample Obtained from Crime Scene or
Paternity InvestigationBiology
DNAExtraction
DNAExtraction
DNAQuantitation
DNAQuantitation
PCR Amplificationof Multiple STR
markers
PCR Amplificationof Multiple STR
markers
Technology
Separation and Detection of PCR Products(STR Alleles)
Sample Genotype
Determination
Genetics
Comparison of Sample Genotype to Other
Sample Results
Comparison of Sample Genotype to Other
Sample Results
If match occurs, comparison of DNA profile to population databases
If match occurs, comparison of DNA profile to population databases
Generation of Case Report with Probability
of Random Match
Generation of Case Report with Probability
of Random Match
Steps in Forensic DNA Typing
Make copies (extend primers)
Starting DNA Template
5’
5’
3’
3’
5’
5’
3’
3’
Add primers (anneal)
5’3’
3’5’
Forward primer
Reverse primer
Polymerase Chain Reaction (PCR)
Separate strands
(denature)
5’
5’3’
3’
PCR is a method of amplifying a specific region of the genome – go from 1 to over a billion copies in about several hours
Multiplex PCR• Over 10 Markers Can Be
Copied at Once• Sensitivities to levels less
than 1 ng of DNA• Ability to Handle Mixtures
and Degraded Samples• Different Fluorescent Dyes
Used to Distinguish STR Alleles with Overlapping Size Ranges
AMEL
D3S1358TH01
TPOX
D2S1338
D19S433
FGA
D21S11
D18S51
CSF1PO
D16S539
D7S820
D13S317
D5S818
VWA
D8S1179
1 integrated analysis vs. 16 separate runs1 integrated analysis vs. 16 separate runs
Information is tied together with multiplex PCR and data analysis
AmpFlSTR® Identifiler™ (Applied Biosystems)
ABI 3100 16-capillary array
ABI 310 single capillary
Capillary Electrophoresis Instrumentation
DAD
MOM
CHILD
Genetic Inheritance Pattern of DNA Profiles
Family Inheritance of STR Alleles (D13S317)
Father
Child #1
Child #2
Child #3
Mother
PCR product size (bp)
11 14
11
12 14
8 14
12
128
Me
PATERNITY TESTING
Results of DNA Tests Impact Families
Results of DNA Tests Impact Families
Family Inheritance of STR Alleles (D13S317)
Father
Child #1
Child #2
Child #3
Mother
PCR product size (bp)
11 14
11
12 14
8 14
12
128
Amanda
Marshall
Katy
Me
My Wife
PATERNITY TESTING
Crime Scene - Two Suspects
Suspect 1
Suspect 2
Evidence
D3 vWA FGA S1 14,15 17,18 23,24S2 15,18 17,19 23.2,24E 15,18 17,19 23.2,24
Main Tasks
Paternity TestingIndividual Identification
Conclusion
Forensic DNA Typing
The genome of each individual is unique (with the exception of identical twins)
Probe subsets of genetic variation in order to differentiate between individuals
DNA typing must be done efficiently and reproducibly (information must hold up in court)
Reference
• Forensic DNA Typing , John Butler • NIST website: http://www.cstl.nist.gov/biotech/strbase
STRBase
Thank you for your attention!Thank you for your attention!谢谢各位同学谢谢各位同学 !!